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Novel applications of shotgun phage display

Rosander, Anna (2004). Novel applications of shotgun phage display. Diss. (sammanfattning/summary) Uppsala : Sveriges lantbruksuniv., Acta Universitatis agriculturae Sueciae. Agraria, 1401-6249 ; 450
ISBN 91-576-6461-7
[Doctoral thesis]

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Abstract

In a shotgun phage display library, theoretically, the entire proteome of a bacterium is represented. Phages displaying specific polypeptides can be isolated by affinity selection, while the corresponding gene remains physically linked to the gene product. The overall objective of the study in this thesis was to explore the shotgun phage display technique in new areas. Initially, it was used to study interactions between Staphylococcus aureus and an in vivo coated biomaterial. It was shown to be well suited for the identification of bacterial proteins that bind to ex vivo central venous catheters. Several known interactions were detected, but it was also found that β2-glycoprotein I (β2-GPI) is deposited on this type of biomaterial – a finding that is of interest both for the adherence of S. aureus, but perhaps also in view of the occurrence of autoantibodies in certain autoimmune diseases. Further, it is of interest to identify the subset of extracellular proteins in a bacterium since they are involved in important functions like pathogenesis and symbiosis. A method that allows for the rapid and general isolation of extracellular proteins is desirable, and may prove particularly useful when applied to bacteria for which the genome sequences are not known. For this purpose, a specialised phage display method was developed to isolate extracellular proteins by virtue of the presence of signal peptides (SS phage display). It was successfully applied to S. aureus and, on a larger scale, to the symbiotic bacterium Bradyrhizobium japonicum. In elaboration of the SS phage display method, an inducible antisense RNA system was incorporated to enable gene silencing of the isolated genes. A tetracycline-regulated promoter was inserted in such a way, that an antisense RNA covering the cloned gene could be expressed. The new element was shown to be compatible with the properties of SS phage display, and to promote gene expression upon induction on both the transcriptional and translational level. However, screening for clones affected by the induction of antisense RNA transcription was unsuccessful, and further developments of the system are required to improve the efficiency of this attractive application.

Authors/Creators:Rosander, Anna
Title:Novel applications of shotgun phage display
Year of publishing :April 2004
Volume:450
Number of Pages:46
Papers/manuscripts:
NumberReferences
ALLI. Rosander, A., Bjerketorp, J., Frykberg, L. & Jacobsson, K. 2002. Phage display as a novel screening method to identify extracellular proteins. Journal of Microbiological Methods 51, 43-55. II. Rosander, A., Frykberg, L., Ausmees, N. & Müller, P. 2003. Identification of extracytoplasmic proteins in Bradyrhizobium japonicum using phage display. Molecular Plant-Microbe Interactions 16, 727-737. III. Rosander, A., Jacobsson, K. & Frykberg, L. Shotgun phage display in combination with an inducible antisense RNA system. Manuscript. IV. Bjerketorp, J., Rosander, A., Nilsson, M., Jacobsson, K. & Frykberg, L. Sorting of a Staphylococcus aureus phage display library against ex vivo biomaterial. Submitted. V. Jacobsson, K., Rosander, A., Bjerketorp, J. & Frykberg, L. 2003. Shotgun phage display - selection for bacterial receptins or other exported proteins. Biological Procedures Online 5, 123-135.
Place of Publication:Uppsala
ISBN for printed version:91-576-6461-7
ISSN:1401-6249
Language:English
Publication Type:Doctoral thesis
Full Text Status:Public
Agris subject categories.:X Agricola extesions > X30 Life sciences
Subjects:Not in use, please see Agris categories
Agrovoc terms:proteins, microbiological analysis
Keywords:Shotgun phage display, extracellular proteins, protein export, gene regulation, antisense RNA, biomaterial, Staphylococcus aureus, Bradyrhizobium japonicum, Escherichia coli
URN:NBN:urn:nbn:se:slu:epsilon-251
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-251
ID Code:511
Department:(NL, NJ) > Dept. of Microbiology (until 161231)
Deposited By: Anna Rosander
Deposited On:03 May 2004 00:00
Metadata Last Modified:02 Dec 2014 10:05

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